The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), phagocytosis, inflammatory mediator release, clearance of antigen, and antibody half-life (reviewed in Daëron, Annu. Rev. Immunol., 1997; 15:203-234; Ward and Ghetie, Therapeutic Immunol., 1995; 2:77-94; Ravetch and Kinet, Annu. Rev. Immunol., 1991; 9:457-492, each of which is incorporated herein by reference).
Antibody constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Of the various human immunoglobulin classes, human IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4.
Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. The Fc region is central to the effector functions of antibodies. The crystal structure of the human IgG Fc region has been determined (Deisenhofer, Biochemistry, 20:2361-2370 (1981), which is incorporated herein by reference). In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys 226.
The effector functions mediated by the antibody Fc region can be divided into two categories: (1) effector functions that operate after the binding of antibody to an antigen; these functions involve the participation of the complement cascade or Fc receptor (FcR)-bearing cells; and (2) effector functions that operate independently of antigen binding; these functions confer persistence in the circulation and the ability to be transferred across cellular barriers by transcytosis (Ward and Ghetie, Therapeutic Immunology, 1995, 2:77-94, which is incorporated herein by reference).
While binding of an antibody to the requisite antigen has a neutralizing effect that might prevent the binding of a foreign antigen to its endogenous target (e.g., receptor or ligand), efficient effector functions are also required for removing and/or destroying foreign antigens.
Several antibody effector functions are mediated by Fc receptors (FcRs), which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcγR, for IgE as FcεR, for IgA as FcαR and so on. Surface receptors for immunoglobulin G are present in two distinct classes—those that activate cells upon their crosslinking (“activation FcRs”) and those that inhibit activation upon co-engagement (“inhibitory FcRs”). Activation FcRs for IgG require the presence of the Immune Tyrosine Activation Motif (ITAM) to mediate cellular activation. This 19 amino acid sequence, found in the cytoplasmic tail of the receptors or their associated subunits, interacts with src and syk families oftyrosine kinases sequentially. Upon crosslinking of an activation FcγR by an immune complex, ITAM sequences trigger the activation of these tyrosine kinases, which in turn activate a variety of cellular mediators, like PI3K, PLCγ and Tec kinases. The net result of these activation steps is to increase intracellular calcium release from the endoplasmic reticulum stores and opening of the capacitance coupled calcium channel to generate a sustained calcium response. These calcium fluxes are critical for the exocytosis of granular contents, stimulation of phagocytosis and ADCC responses and activation of specific nuclear transcription factors. Opposing these activation responses is the inhibitory FcγR. Inhibitory signaling is dependent on a 13 amino acid cytoplasmic sequence called the Immune Tyrosine Inhibitory Motif (ITIM). Upon co-ligation of an ITAM containing receptor to the inhibitory FcγR, a critical tyrosine residue in the ITIM becomes phosphorylated, leading to the recruitment of a specific SH2-containing inositol polyphosphate 5 phosphatase called SHIP. SHIP catalyzes the hydrolysis of the membrane inositol lipid, PIP3, thereby preventing activation of PLCγ and Tec kinases and abrogating the sustained calcium flux mediated by the influx of calcium through the capacitance coupled channel.
Three subclasses of FcγR have been identified: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Because each FcγR subclass is encoded by two or three genes, and alternative RNA splicing leads to multiple transcripts, a broad diversity in FcγR isoforms exists. The three genes encoding the FcγRI subclass (FcγRIA, FcγRIB and FcγRIC) are clustered in region 1q21.1 of the long arm of chromosome 1; the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encoding FcγRIII (FcγRIIIA and FcγRIIIB) are all clustered in region 1q22.
The mouse expresses two activation FcγRs, FcRI and FcRIII, oligomeric surface receptors with a ligand binding α subunit and an ITAM containing γ subunit. The inhibitory receptor is FcγRIIB, a single chain receptor with an ITIM sequence found in the cytoplasmic tail of the ligand binding a chain. FcRIIB and FcRIII bind monomeric IgG with an affinity constant of 1×106; hence, under physiological conditions they do not bind monomeric IgG, but interact with multimeric IgG immune complexes with low affinity and high avidity. FcRIII is the physiologically important activation FcR for mediating inflammatory disease triggered by cytotoxic antibodies or pathogenic immune complexes. FcRIII is expressed on NK cells, macrophages, mast cells and neutrophils in the mouse. It is not found on B cells, T cells or circulating monocytes. FcRIIB is found on B cells, macrophages, mast cells, neutrophils. It is not found on T cells or NK cells. FcRII and III have greater than 90% sequence identity in their extracellular, ligand binding domain.
The situation in the human is more complex. There are two low-affinity activation FcRs for IgG—FcγRIIA and FcγRIIIA. FcγRIIA is a single-chain low affinity receptor for IgG, with an ITAM sequence located in its cytoplasmic tail. It is expressed on macrophages, mast cells, monocytes, neutrophils and some B cells. It is 90% homologous in its extracellular domain to the human inhibitory FcRIIB molecule, which has an ITIM sequence in its cytoplasmic domain, expressed on B cells, macrophages, mast cells, neutrophils, monocytes but not NK cells or T cells. FcRIIIA is an oligomeric activation receptor consisting of a ligand binding α subunit and an ITAM containing γ or ζ subunit. It is expressed on NK cells, macrophages and mast cells. It is not expressed on neutrophils, B cells or T cells. In addition, a receptor with greater than 95% sequence identity in its extracellular domain called FcRIIIB is found on human neutrophils as a GPI-anchored protein. It is capable of binding immune complexes but not activating cells in the absence of association with an ITAM containing receptor like FcRIIA. FcRII and FcRIII are about 70% identical in their ligand binding extracellular domains.
Thus, in the human, IgG cytotoxic antibodies interact with four distinct low-affinity receptors—two of which are capable of activating cellular responses, FcRIIA and FcRIIIA, one of which is inhibitory, FcRIIB and one of which will bind IgG complexes but not trigger cellular responses, FcRIIIB. Macrophages express FcRIIA, FcRIIB and FcRIIIA, neutrophils express FcRIIA, FcRIIB and FcRIIIB, while NK cells express only FcRIIIA. The efficacy of a therapeutic anti-tumor antibody will thus depend on the specific interactions with activation, inhibition and inert low-affinity FcRs, differentially expressed on distinct cell types.
Well-defined tumor models for which therapeutic anti-tumor antibodies have been developed are known. For example, antibodies directed against the HER2/neu growth factor receptor prevent the growth of breast carcinoma cells in vitro and in vivo. Similarly, antibodies directed to the CD20 antigen on B cells arrests the growth of non-Hodgkin's lymphoma (Taji, H. et al., Jpn. J. Cancer Res., 1998, 89:748, which is incorporated herein by reference). These antibodies were developed based on their ability to interfere with tumor cell growth in vitro and are representative of a class which include those with specificities for the EGF receptor (Masul, H. et al., J. Cancer Res., 1986, 46:5592, which is incorporated herein by reference), IL-2R (Waldmann, T. A., Ann. Oncol., 1994, 5 Supp. 1:13-7, which is incorporated herein by reference) and others (Tutt, A. L. et al., J. Immunol., 1998, 161:3176, which is incorporated herein by reference). HERCEPTIN®, a humanized antibody specific for the cellular proto-oncogene p185HER-2/neu (Pegram, M. D. et al., J. Clin. Oncol. 1998, 16:2659; Carter, P. et al., Proc. Natl. Acad. Sci. USA, 1992, 89:4285-4289, each of which is incorporated herein by reference), and RITUXAN®, a chimeric antibody specific for the B cell marker CD20 (Leget, G. A. and Czuczman, M. S., Curr. Opin. Oncol., 1998, 10:548-51, which is incorporated herein by reference), are approved for the treatment of HER-2 positive breast cancer and B cell lymphoma, respectively. A number of in vitro studies indicated that the critical mechanism responsible for the anti-tumor activities of HERCEPTIN® and its mouse parent molecule 4D5 are due to receptor-ligand blockade (Kopreski, M. et al., Anticancer Res., 1996, 16:433-6; Lewis, G. D. et al., Cancer Immunol. Immunother., 1993, 37:255-63, each of which is incorporated herein by reference), while other in vitro studies have suggested that activities such as antibody dependent cellular cytotoxicity (ADCC) may be of importance (Carter, 1992, supra; Lewis, G. D. et al., Cancer Immunol. Immunother., 1993, 37:255-63, which is incorporated herein by reference). In vitro studies with RITUXAN® and its murine parent 2B8 have suggested a direct pro-apoptotic activity may be associated with this antibody (Shan, D. et al., Blood, 1998, 91:1644-52, which is incorporated herein by reference).
Thus, multiple mechanisms have been proposed for the ability of anti-tumor antibodies to mediate their effects in vivo, including extended half-life, blockade of signaling pathways, activation of apoptosis and effector cell mediated cytotoxicity. The elucidation of a mechanism that enhances the ability of anti-tumor antibodies to effectively treat tumors is highly desirable.